Osteoimplant and method of making same

ABSTRACT

An osteoimplant is provided which comprises a coherent aggregate of elongate bone particles, the osteoimplant possessing predetermined dimensions and shape. The osteoimplant is highly absorbent and sponge-like in nature. Also provided herein are a method of fabricating the osteoimplant and a method of repairing and/or treating bone defects utilizing the osteoimplant.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US01/22853, filed Jul. 19, 2001, and claims the 35 U.S.C. §119(e) benefit of provisional applications No. 60/219,198, filed Jul. 19,2000 and No. 60/288,212 filed May 2, 2001. The entire contents ofaforesaid applications PCT/US01/22853, 60/219,198 and 60/288,212 areincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an osteoimplant of predetermined dimensionsand shape made up of a coherent aggregate of elongate bone particles andto a method for making the osteoimplant. Among its other applications,the osteoimplant can be fashioned as a plug for insertion in a space orcavity within an implant used in an orthopedic procedure, e.g., anintervertebral spacer employed in spinal fusion, or for insertion in acavity associated with a relatively well-defined bone defect, e.g., anextraction socket, a bore hole, etc.

2. Description of the Related Art

Shaped or cut bone elements have been used extensively to treat variousmedical problems in human and animal orthopedic surgical practice. Theuse of such bone has also extended to the fields of, e.g., cosmetic andreconstructive surgery, dental reconstructive surgery, podiatry,orthopaedics, neurosurgery and other medical fields involving hardtissue. The use of autograft bone (where the patient provides thesource), allograft bone (where another individual of the same speciesprovides the source) or xenograft bone (where another individual of adifferent species provides the source) is well known in both human andveterinary medicine. In particular, transplanted bone is known toprovide support, promote healing, fill bony cavities, separate bonyelements (such as vertebral bodies), promote fusion (where bones areinduced to grow together into a single, solid unit) or stabilize thesites of fractures. More recently, processed bone has been developedinto shapes for use in new surgical applications or as new materials forimplants that were historically based on non-biologically derivedmaterials.

A particularly advantageous application of shaped or cut bone elements,particularly those derived from allograft bone, is that involving thefusion of adjacent vertebral bodies where there has been damage orinjury to the intervertebral disc.

Intervertebral discs, located between the end plates of adjacentvertebrae, stabilize the spine, distribute forces between vertebrae andcushion vertebral bodies. A normal intervertebral disc includes asemi-gelatinous component, the nucleus pulpous, which is surrounded andconfined by an outer fibrous ring, the annulus fibrous. In a healthy,spine, the annulus fibrous prevents the nucleus pulpous from protrudingoutside the disc space.

Spinal discs may be displaced or damaged due to trauma, disease, oraging. Disruption of the annulus fibrosis allows the nucleus pulposus toprotrude into the vertebral canal, a condition commonly referred to as aherniated or ruptured disc. The extruded nucleus pulposus may press upona spinal nerve, resulting in nerve damage, pain, numbness, muscleweakness and/or paralysis. Intervertebral discs may also deteriorate dueto the normal aging process or disease. As a disc dehydrates andhardens, the disc space height will be reduced leading to instability ofthe spine, decreased mobility and pain.

Sometimes the only relief from the symptoms of these conditions is adiscectomy or surgical removal of a portion or all of an intervertebraldisc followed by fusion of the adjacent vertebrae. The removal of thedamaged or unhealthy disc will allow the disc space to collapse.Collapse of the disc space can cause instability of the spine, abnormaljoint mechanics, premature development of arthritis or nerve damage inaddition to severe pain. Pain relief via discectomy and arthrodesisrequires preservation of the disc space and eventual fusion of theaffected motion segments.

Bone grafts have been used to fill the intervertebral space to preventdisc space collapse and promote fusion of adjacent vertebrae across thedisc space. In early techniques, bone material was simply implantedbetween adjacent vertebrae, typically at the posterior aspect of thevertebra, and the spinal column was stabilized by way of a plate or rodspanning the affected vertebrae. Once fusion occurred, the hardware usedto maintain the stability of the vertebrae became superfluous and becamea permanent non-functional foreign body. Moreover, the surgicalprocedures employed to implant a rod or plate to stabilize the spinewhile fusion was taking place were frequently lengthy and involved.

It was therefore determined that a better solution to the stabilizationof an excised disc space is to fuse the vertebrae between theirrespective end plates, preferably without the need for anterior orposterior rod or plate. There have been numerous attempts to develop anacceptable intradiscal implant that could be used to replace a damageddisc and maintain the stability of the disc inter space between theadjacent vertebrae, at least until complete arthrodesis is achieved. Theimplant must provide temporary support and allow bone ingrowth. Successof the discectomy and fusion procedure requires the development of acontiguous growth of bone to create a solid mass because the implant maynot withstand the compressive loads on the spine for the life of thepatient.

Fusion cages provide a space for inserting a bone graft between adjacentportions of bone. In time, the bone and bone graft grow together throughor around the fusion cage to fuse the graft and the bone solidlytogether. One current use of fusion cages is to treat a variety ofspinal disorders, including degenerative disc diseases such as Grade Ior II spondylolisthesis of the lumbar spine. Spinal fusion cages(included in the general term, “fusion cages”) are inserted into theintervertebral disc space between two vertebrae for fusing themtogether. They distract (or expand) a collapsed disc space between twovertebrae to stabilize the vertebrae by preventing them from movingrelative to each other.

The typical fusion cage is cylindrical, hollow and threaded.Alternatively, some known fusion cages are unthreaded or made intapered, elliptical, or rectangular shapes. Known fusion cages areconstructed from a variety of materials including titanium alloys,porous tantalum, other metals, allograft bone, or ceramic material. Forexample, U.S. Pat. No. 5,015,247 to Michelson and U.S. Pat. No.5,782,919 to Zdeblick disclose a threaded spinal cage the contents ofwhich are incorporated herein by reference. The cages are hollow and canbe filled with osteogenic material, such as autograft or allograft,prior to insertion into the intervertebral space. Apertures defined inthe cage communicate with the hollow interior to provide a path fortissue growth between the vertebral end plates.

Fusion cages may be used to connect any adjacent portions of bone. Aprimary use is in the lumbar spine. Other sites include the cervical orthoracic segments of the spine. Fusion cages can be inserted in thelumbar spine using an anterior, posterior or lateral approach. Insertionis usually accomplished through a traditional open operation but alaparoscopic or percutaneous insertion technique can also be used.

Spinal fusion cages are typically designed to support vertebrae in theproper geometry during the fusion process and are not intended toprovide a long term, permanent support. Actual bone fusion is theultimate goal. In order to achieve fusion, bone conducting or inducingmaterials such as bone chips, ceramics, marrow, growth factors, etc.,are packed into the cage in order to provide a favorable environment forbony ingrowth. The success of these methods depends to some extent onthe surgeon's skill in packing the cages and retaining the materials inthe cages during and after implantation. It is especially important thatthe filler material be in contact with the surfaces of the vertebralbodies on either side of the cage as well as any autograft material(s)employed at the surgical site. While many materials will function asadequate cage filler materials, the best results are obtained withmaterials that are osteoinductive and not just osteoconductive. Bonegrafting materials for use in osteoimplants are described in U.S. Pat.No. 4,950,296, the contents of which are incorporated by referenceherein.

Osteoconductive materials are ones that guide bone growth but do notstimulate it. Examples are bone chips and ceramics. Osteoinductivematerials actually cause bone to form and result in faster and morecertain healing. Examples of osteoinductive materials include cancellousbone, demineralized bone and various growth factors. The most commonsource of osteoinductive material is the patient's own bone. Typically,in spinal surgery, this is harvested from the iliac crest in the form ofbone chips and marrow. While effective, it causes secondary damage (tothe harvest site) and requires preparation before it can be used.Furthermore, it is somewhat difficult to maintain in place due to itssemi-fluid nature.

Demineralized bone is an alternative to bone chips and marrow as anosteoinductive material. Demineralized bone comes in various formsincluding powder, gels, pastes, fibers and sheets. The more fluid formssuch as powders, gels and pastes are relatively easy to implant at therepair site but difficult to maintain in place. The production of bonepowder for filling osteoimplants is disclosed and incorporated herein byreference to U.S. Pat. No. 5,910,315 et al. The process of preparingshaped materials derived from elongate bone particles is incorporatedherein by reference to U.S. Pat. No. 5,507,813.

In addition, there is the possibility of wasted material anytime astandard material has to be adapted to fill a cage. Therefore, the needremains for an osteoinductive material that can be used to fill therelatively well-defined cavities of, for example, bone fusion devices,extraction sockets, bore holes, etc. and that doesn't require anyspecial tailoring by the surgeon at the time of implantation yet remainswhere placed for periods of time sufficient to allow suitable bonefusion to take place. It would be advantageous if methods of producingsuch a material could be achieved efficiently and accurately by a simpleprocess. The use of such an osteoinductive material at an appropriatesurgical site would provide improved outcome for implant recipients.

U.S. Pat. No. 5,507,813 describes a surgically implantable sheet formedfrom elongate bone particles, optionally those that have beendemineralized. The sheet can further contain biocompatible ingredients,adhesives, fillers, plasticizers, etc. The osteoinductive sheet is rigidand relatively strong when dry and flexible and pliable when wetted orhydrated. These sheets are available under the tradename Grafton®Flex(Osteotech, Inc., Eatontown, N.J., USA). The sheets must bewetted/hydrated prior to use in order to render them useful forimplantation.

U.S. Pat. No. 4,932,973 describes an artificial organic bone matrix withholes or perforations extending into the organic bone matrix. The holesor perforations are indicated to be centers of cartilage and boneinduction following implantation of the bone matrix into living tissue.

U.S. Pat. No. 4,394,370 discloses a one-piece sponge-like bone graftmaterial fabricated from fully demineralized bone powder ormicroparticulate bone, and reconstituted collagen. The sponge-like graftis optionally crosslinked with glutaraldehyde.

Another one-piece porous implant is described in U.S. Pat. No.5,683,459. The implant is made up of a biodegradable polymericmacrostructure composed of chemotactic ground substances such ashyaluronic acid.

SUMMARY OF THE INVENTION

It is an object of the invention to provide an osteoimplant ofpredetermined dimensions and shape derived from elongate bone particles.

It is another object of the invention to provide an osteoimplantfabricated from a coherent aggregate of elongate bone particles whereinthe osteoimplant possesses any one of a wide variety of sizes and shapesnot limited by the original shape of the bone(s) from which the elongatebone particles are obtained.

It is a particular object of the invention to provide a low densityosteoimplant which possesses an open pore structure allowing theosteoimplant to readily absorb fluids such as blood and yet still retainits original shape.

It is another object of the invention to provide an osteoimplantfabricated from elongate bone particles which is flexible when dry andwhich can be implanted while in the dry state.

It is yet another further object of the invention to provide an methodof making an osteoimplant possessing the aforementioned characteristics.

It is still another object of the invention to provide a method oftreating a bone defect which utilizes an osteoimplant possessing theaforementioned characteristics.

Another particular object of the invention is the provision of a plugfor insertion in a cavity of an implant, e.g., an intervertebralimplant, or bone defect site made up of a coherent aggregate of elongatebone particles sized and shaped to substantially fill the cavity of theimplant or bone defect site.

These and other objects of the invention are met by the osteoimplantherein which comprises a coherent aggregate of elongate bone particles,the osteoimplant possessing predetermined dimensions and shape.

The term “osteoimplant” as utilized herein is intended to refer to anydevice or material for implantation that aids or augments bone formationor healing. Osteoimplants are often applied at a bone defect site, e.g.,one resulting from injury, defect brought about during the course ofsurgery, infection, malignancy or developmental malformation. Therefore,such “osteoimplants” are envisioned as being suitably sized and shapedas required for use in a wide variety of orthopedic, neurosurgical, andoral and maxillofacial surgical procedures such as the repair of simpleand compound fractures and non-unions, external and internal fixations,joint reconstructions such as arthrodesis, general arthroplasty, deficitfilling, discectomy, laminectomy, anterior cervical and thoracicoperations, spinal fusions, etc. Therefore, the osteoimplants utilizedherein are intended for implantation at a bony site and are made of anybiocompatible material(s), e.g., bone or bone particles, biocompatiblesynthetic materials, combinations thereof, etc, and may be designed foreither animal or human use. Specifically, the osteoimplant suitable foruse according to the disclosure herein will be any osteoimplantcontaining at least one defined cavity without limitation to theparticular material(s) the osteoimplant is made of or the size or shapeof the cavity. The term “biocompatible” and expressions of like importshall be understood to mean the absence of stimulation of anunacceptable biological response to an implant and is distinguished froma mild, transient inflammation and/or granulation response which canaccompany implantation of most foreign objects into a living organismand is also associated with the normal healing response. Materialsuseful to the invention herein shall be biocompatible if, at the time ofimplantation, they are present in a sufficiently small concentrationsuch that the above-defined condition is achieved.

The term “particle” as utilized herein is intended to include bonepieces of all shapes, sizes, thickness and configuration such as fibers,threads, narrow strips, thin sheets, clips, shards, powders, etc., thatposses regular, irregular or random geometries. It should be understoodthat some variation in dimension will occur in the production of theparticles of this invention and particles demonstrating such variabilityin dimensions are within the scope in this invention. Particles usefulherein can be homogenous, heterogeneous, and can include mixtures ofhuman, xenogenic and/or transgenic material.

The term “human” as utilized herein in reference to suitable sources ofimplantable materials refers to autograft bone which is taken from atleast one site in the graftee and implanted in another site of thegraftee as well as allograft bone which is bone taken from a donor otherthan the graftee.

The term “autograft” as utilized herein refers to tissue that isextracted from the intended recipient of the implant.

The term “allograft” as utilized herein refers to tissue intended forimplantation that is taken from a different member of the same speciesas the intended recipient.

The term “xenogenic” as utilized herein refers to material intended forimplantation obtained from a donor source of a different species thanthe intended recipient. For example, when the implant is intended foruse in an animal such as a horse (equine), xenogenic tissue of, e.g.,bovine, porcine, caprine, etc., origin may be suitable.

The term “transgenic” as utilized herein refers to tissue intended forimplantation that is obtained from an organism that has been geneticallymodified to contain within its genome certain genetic sequences obtainedfrom the genome of a different species. The different species is usuallythe same species as the intended implant recipient but such limitationis merely included by way of example and is not intended to limit thedisclosure here in anyway whatsoever.

The expressions “whole bone” and “substantially fully mineralized bone”refer to bone containing its full or substantially full, originalmineral content.

The expression “demineralized bone” includes bone that has beenpartially, fully, segmentally or superficially (surface) demineralized.

The expression “substantially fully demineralized bone” as utilizedherein refers to bone containing less than about 8% of its originalmineral context.

The term “osteogenic” as applied to the bone plug and/or elongate boneparticle composition thereof shall be understood as referring to theability of an osteoimplant to facilitate or accelerate the growth of newbone tissue by one or more mechanisms such as osteogenesis,osteoconduction and/or osteoinduction.

The term “osteoinduction” shall be understood to refer to the mechanismby which a substance recruits cells from the host that have thepotential for forming new bone and repairing bone tissue. Mostosteoinductive materials can stimulate the formation of ectopic bone insoft tissue.

The term “osteoconduction” shall be understood to refer to the mechanismby which a non-osteoinductive substance serves as a suitable template orsubstrate along which bone may grow.

The term “osteogenesis” shall be understood to refer to cell-mediatedbone formation.

The term “device” as utilized herein is intended to refer to anyosteoimplant that is manufactured predominately of non-bone materials.Such devices are typically made of those materials commonly used in themanufacture of biocompatible implants, e.g., biocompatible metals suchas surgical Bioglass®, biocompatible polymeric materials, e.g.,polylactic acid, polytetrafluoroethylene, etc., or any other suitablebiocompatible non-bone material.

The term “plug” as utilized herein refers to a formed material ofpredetermined size and shape prepared according to the descriptionherein from a coherent aggregate of elongate bone particles.

The term “cavity” as utilized herein should be understood in thebroadest sense possible. Therefore, such term is intended herein torefer to any relatively well-defined space or recess in some otherosteoimplant (hereinafter referred to an “implant” to differentiate fromthe osteoimplant of the invention) or bone defect site. The term cavityis intended to encompass regions contained substantially within theimplant, regions defined by an area or portion of the implant, as wellas the regions defined by the placement of adjacent implants and regionsdefined by relatively well-defined bone defects such as extractionsockets, bore holes, etc. Therefore, the term “cavity” as utilizedherein broadly includes any relatively well-defined defect at a surgicalsite.

The plug embodiment of the osteoimplant of the invention is configuredto fit within the cavities of commercially available implants, e.g.,spinal cages, and relatively well-defined cavities at a surgical site.The plugs are engineered to fit efficiently, are easy to insert and tendto remain where placed. An additional advantage of the plug is that inmany of its configurations, it swells somewhat upon contact with bodyfluids thus ensuring good bone contact at the implant site even wherethe site is irregularly shaped. The plug, when made from demineralizedbone, is generally osteoinductive and the fiber-like shape of the boneparticles further provides a favorable scaffold for bone cell growththus potentially combining osteoconductive and osteoinductiveproperties.

The plug can be formed in a range of shapes and sizes that fitcommercially available osteoimplants, e.g., spinal cages, and relativelywell-defined bone defects. Some designs of cages may require two or moreshaped plugs. The present invention also provides efficient methods formanufacturing the preformed bone plug and methods of using the preformedbone plug for the surgical treatment and/or repair of a bone defectsite.

The term “shaped” as applied to the aggregate of elongate bone particlesherein refers to a determined or regular form or configuration incontrast to an indeterminate or vague form or configuration (as in thecase of a lump or other solid matrix of no special form) and ischaracteristic of such materials as sheets, plates, disks, cones, pins,screws, tubes, teeth, bones, portion of bone, wedges, cylinders,threaded cylinders, and the like, as well as more complex geometricconfigurations.

The term “coherent” as applied to the aggregate of elongate boneparticles refers to the ability of the bone particles to adhere to eachother either, e.g., by entanglement, or by the use of a biocompatiblebinder or adhesive.

The expression “three-dimensional” refers to the ability of the coherentaggregate of elongate bone particles to assume any desired shape andsize.

The expression “open pore structure” as it applies to the coherentaggregate of elongate bone particles constituting one embodiment ofosteoimplant herein shall be understood as referring to the low density,absorbent, sponge-like nature of the osteoimplant in which there are aplurality of accessible pores or openings which are present throughoutthe entire volume of the aggregate.

The term “incorporation” utilized herein refers to the biologicalmechanism whereby host tissue gradually replaces the osteoimplant of theinvention with native host bone tissue. This phenomenon is also known inthe scientific literature as “bone remodeling” or “cellular basedremodeling” and “wound healing response”. Therefore, the term“incorporation” utilized herein shall be understood as embracing what isconveyed to those skilled in the art by the foregoing expressions.

The expression “further treatment” as utilized herein refers toprocedures such as, e.g., lyophilizing, cross-linking treatment,re-mineralization, sterilization, etc., performed either before, duringor after the step of making the osteoimplant as well as post-processingprocedures such as, e.g., machining, laser etching, welding, assemblingof parts, cutting, milling, reactive etching, etc.

Another particularly useful embodiment of the invention herein is anosteoimplant provided as a coherent aggregate, or matrix, of elongatebone particles possessing an open pore structure and a bulk bone densityof less than about 0.3 g/cm³. The open pore structure of the aggregaterenders the osteoimplant highly absorbent of surrounding liquids. Theosteoimplant formed from the aggregate is flexible when dry (i.e., whencontaining less than about 5 weight percent water) and does not requiretime consuming rehydration prior to implantation. It can assume anydesired shape and/or configuration and can be cut to the desireddimensions, e.g., with surgical scissors, before and/or after theaggregate has absorbed fluid. Even in the wetted/hydrated state, theosteoimplant will maintain its original shape and coherency and can bereadily handled by the medical practitioner.

Osteoinductivity can be conveniently quantified as the amount of boneformed in an ectopic site in an athymic nude rat. Scores are rated 0 to4. The osteoimplants of the invention exhibit osteoinductivities of atleast 2, typically greater than 3, when measured in an athymic rat assayas described in Edwards J T, Diegmann M H, Scarborough N L,Osteoinduction of Human Demineralized Bone: Characterization in anAnimal Model, Clin. Orthop. Rel. Res. 357:219 228 (1998).

The osteoimplant of the invention can be combined with a wide variety ofbiocompatible substances which can be introduced into the porous matrixof the osteoimplant and/or into large cavities, depressions, and thelike, produced in the osteoimplant. Thus, the implant herein functionsas a highly effective carrier and/or delivery vehicle for bone-growthinducing and/or otherwise medically useful substances.

Further provided herein is a method of fabricating the osteoimplantherein which comprises providing a quantity of elongate demineralizedbone particles, mixing the elongate demineralized bone particles with anaqueous wetting agent to provide a fluid composition preferablycontaining from about 5 to about 40 volume percent swollen, hydratedbone particles, placing the liquid composition in a mold, and removing asufficient amount of aqueous wetting agent, e.g., by heating the fluidcomposition in the substantial absence of pressure at elevatedtemperature, to provide an osteoimplant comprising a shaped, coherentaggregate, or matrix, of elongate bone particles, preferably one of openpore structure and possessing a bulk bone density of less than about 0.3g/cm³.

Further provided in accordance with the invention is a method ofrepairing and/or treating bone comprising implanting at a bone repairsite an osteoimplant which comprises a shaped and dimensioned coherentaggregate of elongate bone particles, preferably one of open porestructure and possessing a bulk bone density of less than about 0.3g/cm³.

The osteoimplant of the invention can be readily applied to virtuallyany bone repair site in the body and can be utilized alone or incombination with one or more adjunct medical devices and/or procedures.The osteoimplant of the invention finds particular utility in the areasof dental reconstructive surgery and spinal fusion where substantialamounts of body fluid, e.g., saliva and/or blood, are frequentlyencountered or where autograft (e.g., local bone, marrow or iliac crest,etc.) is incorporated in the osteoimplant. The unique ability of theosteoimplant to absorb body fluids and still retain its original shaperepresents a significant advance in the medical field.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1a-1i are non-limiting representations of particular sizes andshapes of osteoimplants prepared and used as described herein;

FIG. 2-2 a are non-limiting representations of an intervertebralimplant, specifically, a diaphyseal ring, containing a plug inaccordance with the invention;

FIG. 3 is a non-limiting representation of a fusion cage whose voidspace is filled with a plug in accordance with the invention;

FIG. 4 is a photograph of an osteoimplant produced in accordance withthe present invention possessing a preformed cavity or depression; and,

FIG. 5 illustrates a mold which can be utilized in the fabrication of anosteoimplant such as that shown in FIG. 4.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The composition of the osteoimplant herein can be made up of from about5 to about 100% fully demineralized and/or demineralized elongate boneparticles. At least about 50 weight percent, more preferably at leastabout 60 weight percent, and most preferably at least about 90 weightpercent of the bone particles present in the osteoimplant herein are ofthe elongate variety. Any non-elongate bone particles that areoptionally included in the osteoimplant can possess a wide range ofdimensions, e.g., powders, chips, etc. The elongate bone particles forma coherent aggregate, or matrix, which imparts porosity and absorbencyto the osteoimplant.

The bone component of the osteoimplant can be obtained from cortical,cancellous, and/or corticocancellous allogenic, xenogenic or transgenicbone tissue. In general, allogenic bone tissue is preferred as thesource of the bone component. The bone component can be fullymineralized or partially or fully demineralized. Porcine and bovinebones are particularly advantageous types of xenogenic bone tissue thatcan be used individually or in combination as sources for the boneparticles although of course other xenogenic or transgenic bone tissuescan also be used. Combinations of fully mineralized and demineralizedbone can also be used.

The bone particles employed in the fabrication of the osteoimplant ofthis invention are generally characterized as “elongate”, i.e., theypossess relatively high median length to median thickness ratios. Inoverall appearance, the elongate bone particles can be described asfilaments, fibers, threads, slender or narrow strips, etc. Thus, e.g.,the elongate bone particles can possess a median length of from about0.05 to about 200 mm, preferably from about 1 to about 100 mm, a medianwidth of from about 0.05 to about 50 mm, preferably from about 0.1 toabout 20 mm, and a ratio of median length to median width of from about10:1 to about 2000:1, preferably of from about 20:1 to about 600:1. Ifdesired, the elongate bone particles can be graded into different sizesto reduce or eliminate any less desirable size(s) of particles that maybe present.

The elongate bone particles can be readily obtained by any one ofseveral methods, e.g., by milling or shaving the surface of an entirebone or relatively large section of bone. Employing a milling technique,one can obtain a mass of elongate bone particles containing at leastabout 20 weight percent of bone particles coming within the aforesaidrange of dimensions.

Another procedure for obtaining the elongate bone particles herein,particularly useful for pieces or sections of bone of up to about 100 mmin length, is the bone processing mill described in commonly assignedU.S. Pat. No. 5,607,269. Use of this bone mill results in the productionof long, thin strips which quickly curl lengthwise to providetubular-like elongate bone particles. The elongate bone particles areoptionally subjected to demineralization in accordance with known andconventional procedures in order to reduce their inorganic mineralcontent. Such demineralization can occur prior to or after forming theelongate particles. Demineralization methods remove the inorganicmineral component of bone by employing acid solutions. Such methods arewell known in the art, see for example, Reddi et al., Proc. Nat. Acad.Sci. 69, pp 1601-1605 (1972), incorporated herein by reference. Thestrength of the acid solution, the shape of the bone particles and theduration of the demineralization treatment will determine the extent ofdemineralization. Reference in this regard may be made to Lewandrowskiet al., J. Biomed Materials Res, 31, pp. 365-372 (1996), alsoincorporated herein by reference.

As used herein, the expression “superficially demineralized” refers tobone particles which have undergone surface demineralization as a resultof which they possess one or more regions of surface-exposed collagen.The expression “partially demineralized bone” refers to bone possessingless than its original mineral content but not less than about 8 weightpercent of its original mineral content. As previously stated,“substantially fully demineralized bone” refers to bone containing lessthan about 8 weight percent, and usually less than about 3 weightpercent, of its original mineral content. Mixtures of one or more of theforegoing types of demineralized bone particles can be employed.Moreover, one or more of the foregoing types of demineralized boneparticles can be employed in combination with nondemineralized boneparticles, i.e., bone particles that have not been subjected todemineralization. It will be understood by those skilled in the art thatfully demineralized bone particles yield a more porous mass compared towhole bone or superficially demineralized bone particles.

When prepared in whole or in part from bone particles that are onlysuperficially demineralized or nondemineralized, the osteoimplant willtend to possess a fairly high compression strength, e.g., oneapproaching that of natural bone. Accordingly, when an osteoimplantexhibiting rigidity, e.g., a compression strength of on the order offrom about 5 to about 200 MPa, preferably from about 20 to about 100 MPaand more preferably from about 25 to about 75 MPa, is desired,superficially demineralized bone particles and/or nondemineralized boneparticles are advantageously employed.

In a preferred demineralization procedure, relatively large mineralizedbone piece(s) from which demineralized bone particles are subsequentlyobtained, or fully mineralized bone particles obtained from suchrelatively large mineralized bone piece(s), are subjected to adefatting/disinfecting step which is followed by an aciddemineralization step. A preferred defatting/disinfectant solution is anaqueous solution of ethanol, the ethanol being a good solvent for lipidsand the water being a good hydrophilic carrier to enable the solution topenetrate more deeply into the bone particles. The aqueous ethanolsolution also disinfects the bone by killing microorganisms and viruses.Ordinarily, at least about 10 to 40 percent by weight of water (i.e.,about 60 to about 90 weight percent of defatting agent such as alcohol)should be present in the defatting disinfecting solution to produceoptimal lipid removal and disinfecting within the shortest period oftime. The preferred concentration range of the defatting solution isfrom about 60 to about 85 weight percent alcohol and most preferablyabout 70 weight percent alcohol. Following defatting, the bone particlesare immersed in acid over time to effect their demineralization. Acidswhich can be employed in this step include inorganic acids such ashydrochloric acid and organic acids such as peracetic acid. After acidtreatment, the demineralized bone particles are rinsed with sterilewater for injection to remove residual amounts of acid and thereby raisethe pH. The elongate bone particles used in the manufacture of the plugnaturally entangled or may be mechanically entangled employing, e.g.,the wet laying procedure, akin to a paper-making process, described inaforementioned U.S. Pat. No. 5,507,813 to Dowd et al., to provide asheet-like coherent mass of bone particles which can thereafter beshaped, e.g., by cutting, molding, etc., before or after drying and/orother processing into configurations corresponding to those desired forthe bone plug of this invention.

If desired, the bone particles before or after their being gathered intoa coherent aggregate can be modified in one or more ways, e.g., theirprotein content can be augmented or modified as described in U.S. Pat.Nos. 4,743,259 and 4,902,296, the contents of which are incorporated byreference herein. The elongate bone particles can also be admixed withone or more substances such as binders/fillers, plasticizers,biostatic/biocidal agents, surface active agents, and the like, priorto, during, or after shaping the elongate bone particles into a desiredconfiguration and size. One or more of such substances can be combinedwith the bone particles by soaking or immersing the elongate boneparticles in a solution or dispersion of the desired substance, byphysically admixing the elongate bone particles and the desiredsubstance, co-extrusion of the substance and particles, and the like.

Suitable binders/fillers include cyanoacrylates, epoxy-based compounds,dental resin sealants, dental resin cements, calcium phosphate andcalcium sulfate self-setting cements, glass ionomer cements, polymethylmethacrylate, gelatin-resorcinol-formaldehyde glues, protein andcollagen-based glues, acrylic resins, cellulosics, bioabsorbablepolymers such as polyglycolide, polylactide, glycolide-lactidecopolymers, polycaprolactone, polyanhydrides, polycarbonates,polyorthoesters, polyamino acids, polyarylates, polycyanoacrylates,polyhydroxybutyrate, polyhydroxyvalyrate, polyphosphazenes, andpolyvinylpyrrolidone, carbohydrate polymers, polyiminocarbonates,polypropylene fumarates, polyanhydride esters, polytetrafluoroethylene,hexacryl, Hyaluronic acid, fibrin, fibrin-collagen, polyethylene glycolglues, mucopolysaccharides, mussel adhesive proteins, fatty acids andfatty acid derivatives, etc.

Other suitable biners/fillers include bone powder, demineralized bonepowder, porous calcium phosphate ceramics, hydroxyapatite, tricalciumphosphate, Bioglass® and other calcium phosphate materials, calciumsulfate or calcium carbonate particles, etc.

Suitable plasticizers include liquid poly hydroxy compounds such asglycerol, monoacetin, diacetin, hydrogels, etc.

Suitable biostaticibiocidal agents include antibiotics, povidone,sugars, mucopolysaccharides, etc.

Suitable surface-active agents include the biocompatible nonionic,cationic, anionic and amphoteric surfactants. It will be understood bythose skilled in the art that the foregoing list is not intended to beexhaustive and that other materials may be admixed with bone particleswithin the practice of the disclosure herein such as disclosed in U.S.Pat. No. 5,073,373, the contents of which are incorporated by referenceherein.

Any of a variety of bioactive substances can be incorporated in, orassociated with, the bone particles before, during or after fabricationof the osteoimplant. Thus, one or more of such substances can becombined with the elongate bone particles by soaking or immersing themin a solution or dispersion of the desired substance(s). Bioactivesubstances include physiologically or pharmacologically activesubstances that act locally or systemically in the host.

Representative classes of bioactive factors which can be readilycombined with the bone particles include, e.g., trophic factors,analgesics, anti-cancer agents, vaccines, adjuvants, antibodies,neuroleptics, genes and genetic elements for transfection includingviral vectors for gene therapy, cells or cellular components, etc. Alist of more specific examples would therefore include, collagen,insoluble collagen derivatives, etc., and soluble solids and/or liquidsdissolved therein, e.g., antiviricides, particularly those effectiveagainst HIV and hepatitis; antimicrobials and/or antibiotics such aserythromycin, bacitracin, neomycin, penicillin, polymicin B,tetracyclines, biomycin, chloromycetin, and streptomycins,cephalosporins, ampicillin, azactam, tobramycin, clindamycin andgentamicin, etc.; biocidal/biostatic sugars such as dextran, glucose,etc.; amino acids, peptides, vitamins, inorganic elements, co-factorsfor protein synthesis; hormones; endocrine tissue or tissue fragments,synthesizers; enzymes such as collagenase, peptidases, oxidases, etc.,polymer cell scaffolds with parenchymal cells, angiogenic drugs andpolymeric carriers containing such drugs; collagen lattices; antigenicagents; cytoskeletal agents; cartilage fragments, modified living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells, naturalextracts, genetically engineered living cells or otherwise modifiedliving cells, DNA delivered by plasmid or viral vectors, genes orgenetic elements, tissue transplants, demineralized bone powder,autogenous tissues such as blood, serum, soft tissue, bone marrow, etc.;bioadhesives; non-collagenous proteins such as osteoponfin, osteonectin,bone sialo protein, laminin, fibrinogen, vitronectin, thrombospondin,proteoglycans, decorin, beta glycan, biglycan, aggrecan, versican,tenascin, matrix gla protein, hyaluronan, amino acids, amino acidresidues, peptides, bone morphogenic proteins (BMPs); osteoinductivefactor (OIF); fibronectin (FN); endothelial cell growth factor (ECGF);cementum attachment extracts (CAE); ketanserin; human growth hormone(HGH); animal growth hormones; epidermal growth factor (EGF);interleukin-1 (IL-1); human alpha thrombin; transforming growth factor(TGF-beta); insulin-like growth factor (IGF-1) (IGF-2); platelet derivedgrowth factors (PDGF); fibroblast growth factors (FGF, aFGF, bFGF,etc.); periodontal ligament chemotactic factor (PDLGF); somatotropin;bone digestors; antitumor agents; immuno-suppressants; fatty acids(including polar and non-polar fatty acids); permeation enhancers, e.g.,fatty acid esters such as laureate, myristate and stearate monoesters ofpolyethylene glycol, enamine derivatives, alpha-keto-aldehydes, etc.;and nucleic acids; inorganic elements, inorganic compounds, cofactorsfor protein synthesis, hormones, soluble and insoluble components of theimmune system; soluble and insoluble receptors including truncatedforms; soluble, insoluble and cell surface bound ligands includingtruncated forms; chemokines, bioactive compounds that are endocytosed;endocrine tissue or tissue fragments, growth factor binding proteins,e.g., insulin-like growth factor binding protein (IGFBP-2) (IGFBP-4)(IGFBP-5) (IGFBP-6); angiogenic agents, bone promoters, cytokines,interleukins, genetic material, genes encoding bone promoting actions,cells containing genes encoding bone promoting action; growth hormonessuch as somatotrophin; bone digestors; antitumor agents; cellularattractants and attachment agents; immuno suppressants; bone resorptioninhibitors and stimulators; angiogenic and mitogenic factors; bioactivefactors that inhibit and stimulate secondary messenger molecules; celladhesion molecules, e.g., cell-matrix and cell-cell adhesion molecules;secondary messengers, monoclonal antibodies specific to cell surfacedeterminants on mesenchymal stem cells, clotting factors; externallyexpanded autograft or xenograft cells, nucleic acids and any combinationthereof. The amounts and types of such optionally added substances canvary widely with optimum levels and combinations being readilydetermined in a specific case by routine experimentation.

Various shapes of osteoimplant can be made by using extrusion orinjection molding techniques, compression molds, pre-formed molds inwhich the material can be placed to obtain its final shape, orpre-formed shapes which can be used to cut desired shape from pre-formedmaterial. Further to this, devices can be used that allow introductionof various agents in to the molding devices or treatments of the moldsto assist in the forming of various shapes, such as, but not limited to,cross linking agents or heat or cooling of the molds and/or during theforming process. Employing such procedures, various sizes and shapes ofosteoimplant can be provided such as those illustrated in FIGS. 1a -i.

In one embodiment herein, an osteoimplant as previously described ismade by forming demineralized elongate bone particles into a coherentaggregate and thereafter either cutting the osteoimplant from theaggregate or, preferably, to reduce waste, molding the aggregate into anosteoimplant of the desired size and configuration. To fabricate thecoherent mass of elongate bone particles, a quantity of elongate boneparticles with or without one or more optional materials is mixed with asuitable biocompatible fluid component, e.g., water, organic proteinsolvent, physiological saline, concentrated saline solution, ionicsolution of any kind, aqueous sugar solution, liquid polyhydroxycompound such as glycerol or glycerol ester, hydrogel, etc., or mixturesthereof. The suitable biocompatible fluid can optionally contain one ormore substances such as binder, filler, plasticizer, biostatic/biocidalagent, surface active agent, bioactive substance, etc., as previouslydescribed to form a slurry or paste. Excess fluid is then removed fromthe slurry or paste, e.g., by applying the slurry or paste to a mesh orscreen and draining away excess fluid. Functionally, the biocompatiblefluid provides a coherent aggregate of elongate bone particles whoseconsistency can be described as shape-sustaining but readily deformable,e.g., putty-like.

If desired, the elongate bone particles can be dried, e.g., at fromabout 30° to about 80° C. and preferably from about 40° to about 50° C.,for from about 1 to about 3 hours, and then lyophilized under conditionsthat are well known in the art, e.g., at a shelf temperature of fromabout −20° to about −35° C., a vacuum of from about 150 to about 100mTorr and for a period of time ranging from about 4 to about 48 hours.The drying and lyophilization steps will result in the production of acoherent mass of entangled elongate bone particles that is relativelystrong when dry and flexible when wetted or hydrated.

In another embodiment of the general method described above, thecoherent aggregate of elongate bone particles can be subjected to acompressive force, e.g., of up to about 100,000 psi, during and/or afterthe step of removing excess liquid and/or while thedrained-but-still-wet bone particles are being dried. If desired, thecompressed coherent mass can be lyophilized to provide an especiallystrong and rigid mass.

In yet a further embodiment disclosed herein, the elongate boneparticles, in combination with bone particles possessing othergeometries such as mineralized and demineralized bone powders andpieces, can be combined with a wetting agent as described above toproduce a flowable composition containing from about 5 to about 100%,preferably from about 20 to about 60%, volume percent of bone particlesof all types, the remainder of the composition comprising wetting agent.The wetting agent can optionally comprise one or more biocompatiblecomponents as previously described. The wetting agent will cause thedemineralized elongate bone particles to swell and increase inflexibility. The fluid composition will possess a consistency rangingfrom a slurry or paste to a wet dough, depending on the amount ofwetting agent used. The critical aspect is that the elongate boneparticles be suspended in and evenly distributed throughout the fluidcomposition. This is to be contrasted with the “wet laying” procedure ofcommonly assigned U.S. Pat. No. 5,507,813 in which wetting agent issubstantially removed to produce a dense mat of bone particles.

In this embodiment, the fluid composition is formed by mixing the boneparticles and wetting agent to form a liquid slurry, stirring the slurryfor a suitable period of time sufficient to allow the wetting agent topenetrate the demineralized elongate bone particles, and removing enoughwetting agent, e.g., by draining through a sieve, sufficient to providea fluid composition containing from about 5 to about 25, preferably fromabout 10 to about 15, volume percent bone particles. Substantialmechanical entanglement of the elongate bone particles will occur.Suitable wetting agents include biocompatible liquids and/or hydrogelssuch as previously described. Optionally, the wetting agent can comprisedissolved or admixed therein one or more biocompatible substances suchas previously described.

Preferred wetting agents for forming the wetted mass of bone particlesinclude water, liquid polyhydroxy compounds and their esters, andpolyhydroxy compounds in combination with water and/or surface activeagents. Specific polyhydroxy compounds of the foregoing type includeglycerol and its monoesters and diesters derived from low molecularweight carboxylic acids, e.g., monoacetin and diacetin (respectively,glycerol monoacetate and glycerol diacetate), ethylene glycol,diethylene glycol, triethylene glycol, 1,2-propanediol,trimethylolethane, trimethylolpropane, pentaerythritol, sorbitol,polyethylene glycol, polyoxyalkylenes, e.g., Pluronics®, and the like.The preferred polyhydroxy compounds possess up to about 12 carbon atomsand, where their esters are concerned, are preferably the monoesters anddiesters. Of these, glycerol is especially preferred as it improves thehandling characteristics of the bone particles wetted therewith and isbiocompatible and easily metabolized. Most preferred are solutions ofpolyhydroxy compounds in water, with glycerol/water solutions in weightratios ranging from about 40:60 to about 5:95, respectively, beingespecially preferred. Mixtures of polyhydroxy compounds or esters, e.g.,sorbitol dissolved in glycerol, glycerol combined with monoacetin and/ordiacetin, etc., are also useful.

Where the bone particles have a tendency to quickly or prematurelyseparate or to otherwise settle out from the fluid composition such thatformation of a homogeneous suspension of bone particles in wetting agentis rendered difficult, it can be advantageous to include within thecomposition a suspension aid. Thus, e.g., where the wetting agent iswater and/or glycerol and separation of bone particles occurs to anexcessive extent where a particular application is concerned, athickener such as a solution of polyvinyl alcohol, polyvinylpyrrolidone,cellulosic ester such as hydroxypropyl methylcellulose, carboxymethylcellulose, pectin, xanthan gum, food-grade texturizing agent,gelatin, dextran, collagen, starch, hydrolyzed polyacrylonitrile,hydrolyzed polyacrylamide, polyelectrolyte such as polyacrylic acidsalt, hydrogels, chitosan, other materials that can suspend particles,etc., can be combined with the wetting agent in an amount sufficient tosignificantly improve the suspension-keeping characteristics of thecomposition. Furthermore, suspension aids that generate gas bubblesinside the fluid composition can be employed. The gas bubbles reduce thetendency of the bone particles to settle out and include peroxides andbicarbonate.

As stated previously, the fluid composition is preferably placed in amold which optionally is configured and dimensioned in the shape of thefinal osteoimplant. FIG. 5 depicts mold 10 and lid 20 for mold 10, lid20 possessing protruding indentations 30. The mold can be optionallyconfigured and dimensioned in the shape of the final osteoimplant, e.g.,the osteoimplant shown in FIG. 4. Care must be taken to ensure thatminimal, if any, pressure is applied to the composition in the moldwhich would effect compaction of the elongate bone particles. This is incontrast to the wet-lay procedure described in U.S. Pat. No. 5,507,813.The composition is then dried at a temperature of from about 30° C. toabout 80° C., preferably from about 30° C. to about 40° C. to effectremoval of water and provide a shaped material. Following the dryingstep, the shaped material is dried e.g., freeze-dried, employing a shelftemperature of from about −20° to about −35° C. and a vacuum of fromabout 150 to about 100 mTorr applied for from about 4 to about 48 hours.The resulting shaped material is porous and absorbent and maintains itsshape and cohesiveness upon absorption of fluid. The implant can beeasily cut with scissors in either the dry or rehydrated state.

Alternatively, a slurry of demineralized elongate bone particles can beinjected into a porous tube. The bone particles can be dried andlyophilized in the tube, then removed and cut to length. Examples ofsuitable porous tubes are dialysis tubing, sausage casings, and rigidmetal or plastic tubing perforated with a series of small holes (theholes generally being small enough that a few fibers escape, preferably0.2 mm or less). It is also possible to use rigid tubing with largeholes, and line with another tube that will contain the bone particleslurry such as a flexible dialysis tube. The slurry can be injected intothe tube by any suitable means, for example a disposable plastic syringeor a slurry pump. If a thin, flexible tube is used to form theosteoimplant, the osteoimplant can be cut to length (after drying) whileinside the tube by cutting through both the tube and the materialinside. Alternatively, the dried aggregate elongate bone particles canbe removed from the flexible tube (preferably by cutting the tube away)or from a rigid tube (preferably by pushing the material out of thetube) and then cut. Cutting is facilitated by using a cutting jig orguide similar to a cigar cutter or a small double bladed guillotine typeof device where the blade spacing equals the desired plug length.Osteoimplants that are dried in a porous tube can have a tougher skin ontheir outer circumferential surface due to more rapid water loss fromthe surface than the interior. This can be advantageous in that theosteoimplant will resist insertion and handling forces better. Thethickness and toughness of the skin can be influenced by a combinationof drying conditions and tubing porosity.

Optionally, the bone particles in the osteoimplant can be crosslinked inaccordance employing well-known techniques, e.g., those disclosed inU.S. Pat. No. 6,294,187 the contents which are incorporated by referenceherein. These crosslinking procedures result in the formation ofchemical bonds between the surface-exposed collagen of mutuallycontacting surface-demineralized and/or substantially completelydemineralized elongate bone particles making up, or contained in, theaggregate of naturally or mechanically entangled elongate boneparticles.

Where a mold, e.g., a cylindrical mold, is employed to shape thecoherent mass of bone particles into the osteoimplant of this invention,the walls of the mold can be coated with a slurry or paste containingpartially and/or fully demineralized bone particles followed by additionof a slurry or paste containing non-demineralized and/or superficiallydemineralized bone particles (or vice versa). The resulting moldedosteoimplant contains at least one region, e.g., an outer surface,composed of partially and/or fully demineralized bone particles and atleast one region, e.g., a core, composed of non-demineralized and/orsuperficially demineralized bone particles. In this manner, thedifferential in compressive strength, porosity, osteogenicity and otherproperties between partially and/or fully demineralized bone particleson the one hand and non-demineralized and/or superficially demineralizedbone particles on the other hand can be exploited. For example, wherethe osteoimplant is employed in a load-bearing situation,non-demineralized and/or superficially demineralized bone particles canbe concentrated in that region of the osteoimplant which will besubjected to an applied load at the implant site.

When the osteoimplant of this invention is fashioned as a plug, the plugcan assume a determined or regular form or configuration such as may bedesirable for any specific cavity of a commercially availableosteoimplant, e.g., spinal cages, or any other relatively well-definedcavity at a surgical site, e.g., extraction sockets, bore holes, etc. Ofcourse, the coherent mass can be machined or shaped by any suitablemechanical shaping means. Computerized modeling can, for example, beemployed to provide an intricately-shaped plug which is custom-fitted toa particular cavity of an osteoimplant with great precision. In apreferred embodiment, the plug possesses the configuration of a centralcavity of a commercially available spinal cage.

A plug fabricated in accordance with this invention preferably possessesa bone particle content of at least about 5 to about 100 weight percent,preferably at least about 20 weight percent and more preferably at leastabout 60 weight percent, based on the weight of the entire mass. It willbe understood by those skilled in the art that plugs possessing aputty-like consistency will possess lower amounts of bone particles, ona weight-by-weight basis, compared to plugs which are subjected to thedrying and lyophilizing and/or compression steps described above.

The foregoing plug can be easily inserted into a cavity of an implant orbone defect site. The plug will often tend to swell somewhat uponcontact with irrigation fluids such as those that are commonly usedduring surgical procedures or upon contact with bodily fluids normallypresent at the implant site. This swelling has the advantage ofproviding a tighter fit with the cavity of the osteoimplant or bonedefect thus assuring that the plug will remain in place.

The plug can be used for implantation at a surgical site in a variety ofways and for treatment of a variety of bone defects. In certainembodiments, the osteoimplant can be manufactured to contain the plugprior to its packaging and storage. Alternatively, the plug can beprovided in a form suitable for placement in an osteoimplant at the timeof the implantation of the latter. In yet a further embodiment, the plugdescribed herein is provided in a form suitable for association with oneor more osteoimplants or one or more cavities of an osteoimplant afterthe osteoimplant has been placed at an implant site. In yet a furtherembodiment, the preformed plug described herein is provided in a formsuitable for placement in a relatively well-defined defect site such as,e.g., an extraction socket, bore hole, etc. Of course, any combinationof the above-described embodiments may be used depending upon theintended use and/or specific defect site.

In any of the above-described plug embodiments, the plug can be usedalone, associated with standard rehydrating solutions such as previouslydescribed, associated with autograft tissue such as iliac crest, localbone and/or marrow, blood, plasma or serum obtained at the time ofimplantation or sometime prior to implantation as well as combinationsthereof. In certain embodiments, the plug will be placed at an implantsite in the as-packaged condition. In alternative embodiments, the plugcan be rehydrated just prior to implantation.

As previously stated, the plug form of the osteoimplant herein can beinserted into the cavity of the implant as is or, if desired, it can besuitably compressed prior to insertion into the implant or defect site.When the plug is compressed just prior to its insertion, any excessliquid or moisture will be displaced from the material as the volume ofthe plug is temporarily reduced. In this embodiment, the compressedplug, being smaller than its receiving cavity, is easily inserted intosuch cavity. After relaxation of the applied compressive force, the plugwill substantially return to its original volume thus assuring-itsretention in the cavity.

The foregoing description and embodiments were chosen and described tobest explain the principles of the invention and its practicalapplications, thereby enabling others skilled in the act to best utilizethe invention in its various embodiments and with various modificationsas are suited to the particular use contemplated. Therefore, theforegoing descriptions of the preferred embodiments of the disclosureherein have been presented for purposes of illustration and descriptionand are not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Obviously, many other modifications andvariations are possible in light of the above teachings. For example,the plug can be configured to fit a predetermined defect caused at animplant site, e.g., a bore hole of defined diameter and depth.Optionally, the plug can be contacted with fluids such as blood, plasma,serum, bone marrow, etc. obtained from the recipient immediately priorto implantation. Such modifications are also envisioned as being withinthe scope of the invention herein.

The following examples illustrate the practice of the present inventionand in no way limit the scope of the claims appended hereto.

EXAMPLE 1 Process of Making a Species-Specific Osteoimplant with DefinedDimensions

Species-specific (Rhesus Monkey) long bones were aseptically cleaned.The cortical bone was processed in the bone milling apparatus describedin U.S. Pat. No. 5,607,269 to yield 65 grams of elongate bone particles.The elongate bone particles were placed in a reactor and allowed to soakfor 5-10 minutes in 0.6N HCl plus 20-2000 ppm nonionic surfactantsolution. Following drainage of the HCl/surfactant, 0.6N HCl at 15 mlper gram of total bone was introduced into the reactor along with theelongate bone particles. The reaction proceeded for 40-50 minutes.Following drainage through a sieve, the resulting demineralized elongatebone particles were rinsed three times with sterile, deionized water at15 ml per gram of total bone, being replaced at 15 minute intervals.Following drainage of the water, the bone particles were covered inalcohol and allowed to soak for at least 30 minutes. The alcohol wasthen drained and the bone particles were rinsed with sterile deionizedwater. The bone particles were then contacted with a mixture of 4.5 mlglycerol per gram of dry bone particles and 10.5 ml sterile, deionizedwater per gram of dry bone particles for at least 60 minutes. Excessliquid was drained and the resulting liquid composition containingapproximately 11 (w/v) demineralized elongate bone particles wastransferred to a 11 cm×11 cm mold containing a lid having a plurality ofprotruding indentations such as those depicted in FIG. 4. The dimensionsof the protrusions were specific for the size of the osteoimplantrequired for the Rhesus monkey. The lid was gently placed on the moldsuch that the indentations became immersed into the liquid compositionto exert as little pressure on the composition as possible. The mold wasthen placed in an oven at 46° C. for 4 hours. The composition was thenfrozen overnight at −70° C. and then lyophilized for 48 hours. Followinglyophilization, the mold was disassembled and the formed composition wascut into individual pieces that contained troughs corresponding to thedimensions of the lid protrusions. The resulting pieces had dimensionsof 4.5 cm in length, 2.5 cm in width and about 8 mm in height withtrough dimensions of 3.5 cm in length, 1 cm in width and 4 mm of depth.

The resulting composition was cohesive, flexible, and sponge-like withan obvious continuous three-dimensional structure possessing visibleopen pores. The implant had a defined shape including the indentationsmade by the lid protrusions, did not require rehydration before use, andwas more rapidly hydratable in comparison to Grafton®Flex. The materialretained its shape once wetted with fluids and freezing was not requiredfor storage.

The bulk density of bone in the osteoimplant is based on calculation ofthe defined mold volume used and the amount of demineralized boneparticles used to fill the volume of the mold. In making the compositiondescribed in this example, 12 g demineralized fibers occupied a volumeof 105 cm³. Therefore, the bulk bone density was approximately 0.114 gof bone/cm³. These calculations are approximate as there can be a rangein weights (about 10-20 g) and a range in volumes of about 100-120 cm³(which can be defined by the dimensions of the mold used).

EXAMPLE 2 Evidence of Osteoinduction by Grafton DBM in Non-Human PrimateSpine Fusion

While autogenous iliac crest bone graft remains the “gold standard”,much work continues to identify viable bone graft extenders, enhancers,and substitutes. While several demineralized bone matrix formulationshave been shown to be variably osteoinductive in rodent ectopic boneassays, few have demonstrated efficacy in higher species and morechallenging applications such as posterolateral spine fusion. To date,none have been tested in a non-human primate posterolateral spine fusionmodel which has been previously determined to be extremely challengingwith less than 40% of animals achieving successful fusion withautogenous iliac crest bone graft. The purpose of this example was totest the osteoimplant described in Example 1 for evidence ofosteoinduction and its use as an extender/enhancer for autogenous bonegraft in a non-human primate.

Four skeletally mature rhesus macaques underwent single level lumbarposterolateral arthrodesis through a Wiltse muscle-splitting approachunder general anesthesia. The transverse processes were decorticatedwith an electric burr. Autogenous iliac crest bone graft was harvestedbilaterally through separate fascial incisions. In these four animals,rhesus-specific osteoimplant material (described in Example 1) wasimplanted with the usual autograft (4 g) on one side of the spine andone half the usual autograft (2 g) on the opposite side. Radiographswere taken at intervals until euthanasia at 24 weeks. The lumbar spineswere excised and palpated manually to determine fusion status as fusedor not fused and then underwent CT scanning to visualize the amount ofbone formation. Radiographs and CT scans were evaluated blindly andassessed semi-quantitatively for the area of the fusion mass (3=good,2=fair, 1=poor) and the amount of bridging between the transverseprocesses on each side (0=<25%, 1=25%, 2=50%, 3=75%, 4=100%). Pointswere added for each site in each animal. Three of four monkeys receivingthe osteoimplant plus autograft were graded as fused. Six of eight sitesin the were rated as “good” for area of fusion mass on CT (computertomography) scans. Six of eight sites had at least 50% bridging. Thequality and amount of bone was better in the osteoimplant group and bestwith the 4 g of autograft. Although the assessment of bone formation wassemi-quantitative, given the spectrum of fusions previously obtained inthis model with autograft alone, these data support evidence ofosteoinduction of the osteoimplant in a challenging model. These datasupport the role of this osteoimplant as an osteoinductive graftextender and graft enhancer in rhesus posterolateral spine fusion.

EXAMPLE 3 Implantation of Osteoimplant in a Human Patient to PromoteSpinal Fusion

Human-specific osteoimplant was made in the same manner described inExample 1. However, the mold dimensions and final dimensions of theosteoimplant were altered to adjust to the approximate size required forhuman posterolateral spinal fusion procedure (known by those skilled inthe art). The dimensions of the osteoimplant pieces were approximately5.0 cm in length, 2.5 cm in width and approximately 1 cm in height withtrough dimensions 4 cm in length, 1.5 cm in width and depthapproximately 0.7 cm. The trough design specifically allowed for thesurgeon to fill the center of the osteoimplant with autograft orallograft or both. Autograft is usually obtained from local bone at thesite of the procedure, or marrow, or iliac crest or a combination. Thefluids rapidly dispersed within the osteoimplant hydrating theosteoimplant. The osteoimplant is placed either trough down facing thedecorticated transverse processes or trough facing away from thedecorticated transverse processes to allow blood to be absorbed by thesponge-like nature of the osteoimplant. The osteoimplant remains as athree-dimensional cohesive structure retaining the autograft orallograft or both at the implant site. The surgery then follows usualclosure procedure known to those skilled in the art.

EXAMPLE 4 Evaluation of the Osteoinductive Potential of Example 1

The osteoinductive potential of Example 3 (human-specific osteoimplant)for posterolateral fusion (PLF) was evaluated using the standardheterotopic osteoinductive implant model (see, Edwards J T, Diegmann MH, Scarborough N L, Osteoinduction of human demineralized bone:Characterization in an animal model, Clin Orthop Rel Res 357:219228(1998) which is a modification of Urist M R, Bone formation byautoinduction, Science, 150:893-899 (1965)). Implants are placed in thehind limb, intramuscular sites of athymic rats and evaluatedhistologically after 28 days.

Animal Model

The study was conducted in the athymic (nude) rat to minimize thepotential for a cross species incompatibility response to xenografttissue implants. The hind-limb intramuscular site is ideal for theinitial determination of heterotopic bone induction properties ofimplant materials, as bone is not present in this area.

Implant Placement

The study utilized a singular intramuscular (IM) implantation site ineach hind limb of the animals. Different specimen types were placed inthe sites in a randomized fashion, such that the same animal did nothave the same treatment in both hind limbs. To provide a common positivecontrol over all animals, a single 40 mg sample of rat DBM powder wasplaced intramuscularly over the left pectoralis (LP) muscle on the leftside of each rat. Animals were allowed normal activities followingsurgical procedures. Four samples of each material were used foranalysis.

Procedure

Briefly, rats were anesthetized with a mixture of ketamine (250 mg),xylazine (11 mg), and physiological saline (10 ml). The dosage is 3.6ml/kg body weight administered intraperitoneally. Aseptic surgicalprocedures were carried out in a laminar airflow hood. A 1 cm skinincision was made on each upper hind limb using a lateral approach andthe skin was separated from the muscle by blunt dissection. Asuperficial incision aligned with the muscle fiber plane was made toallow for insertion of the tips of the scissors. Blunt dissection of themuscle to create a pocket and positioning of the rat DBM powder ordevitalized fibers was made using a blunt syringe. In each case, theskin was closed with metal clips.

Rats were euthanized with CO₂ following 28-day implantation time.Implant materials were located by palpitation, retrieved by bluntdissection and cleaned of the surrounding tissue by careful trimming. Anobserver blinded to implant type performed a macroscopic evaluation ofthe implant material. Color, vascularity, hardness and integrity werescored according to the scheme outlined in Table I; the highest scorefor the most robust response would be 1, while a specimen showing littleor no osteoinductive potential would score 0. Experience with this modelhas shown a high correlation between visual observations andhistological observations of DBM implant performance.

Histology

Retrieved materials were fixed in neutral buffered formalin, dehydratedin a series of graded ethanol solutions, embedded in JB-4 (glycolmethacrylate, Polysciences, Inc., Warrington, Pa.) and sectioned.Toluidine blue was used for staining and each material was evaluatedusing a light microscope at magnifications up to 200×.

A numerical score of 0, 1, 2, 3, or 4 was given to grade the extent ofnew bone formation for each explant when examined under the lightmicroscope. Assignment of scores was according to the descriptions givenin Table II below. Histological sections for each explant were scoredindependently by two individuals blinded to treatment groups.

Following histological analysis, average scores were calculated for eachmaterial type or sample group. Based on previous experience with thisanimal model, each group was assigned an assessment of osteoinductivepotential based on the average histological scores. Sample groupsscoring 0 show “no osteoinductive response”; groups scoring up to 2 showa “slight osteoinductive response” and groups scoring 3 or above show a“robust osteoinductive response”.

TABLE I Macroscopic Observation Scoring Guidelines Color White (W) Gray(G) Red (R) Vascularity None (N) Some (S) Robust (R) Hardness Mushy (M)Firm (F) Hard (H) Integrity Diffuse (D) Flat (F) Nodule (N) Score 0 0.51

TABLE II Scoring of Histological Sections Score New Bone Formation 0 Nonew bone 1 Few areas of new bone formation 2 Numerous areas of new boneformation 3 Greater than 50% of nodule involved in new bone formation 4Greater than 75% of nodule involved in new bone formationResults

Histology showed evidence of robust cartilage, bone and marrow formationin the samples. Scores for the individual samples were averaged and themean±SD of the osteoinductive score for 13 individual samples derivedfrom Example 3 was 3.3±0.7. Historically, demineralized bone powderproduces a comparable osteoinductive score of 3.6±0.8 while guanidinehydrochloride extracted samples routinely display lack of inductivity.The foregoing results demonstrate that the osteoimplant of the inventionpossesses excellent osteoinductivity with the additional advantage ofbeing a cohesive three-dimensional, lower density, porous matrix.

EXAMPLE 5

This example illustrates the preparation of a plug in accordance withthe invention which is intended for insertion in the cavity of animplant as shown in FIGS. 2, 2 a and 3, or in the cavity of a bonedefect site.

Mineralized milled and sieved fibers of cortical bone were fullydemineralized with a 30 ml volume of 0.6M HCl/0.025% Triton X-100 pergram of bone at room temperature. The acid was decanted and theremaining fibers were washed with deionized water to remove theremaining acid. The demineralized elongate fibers were soaked in a 70%ethanol and deionized water solution for at least 30 minutes. Theelongate demineralized fibers were washed with deionized water to removethe ethanol solution. The wet elongate demineralized fibers were addedto a glycerol-deionized water solution and mixed well. The fibers andthe glycerol-deionized water solution was allowed to sit for at leastone hour. The elongate demineralized fibers were lightly packed in acylindrical vessel to form a mold with a desired configuration which wascapped to prevent loss of moisture. The capped vessel was heated in anoven at 46 degrees Celsius for a period of from two to six hours. Thecapped vessel was then placed in a freezer for from 6 to 24 hours at −70degrees Celsius. The vessel was then removed from the freezer and thecap was removed. The uncapped vessel was then lyophilized for 48 hours.The lyophilized plug was then removed from the container.

What is claimed is:
 1. An osteoimplant which comprises elongate boneparticles selected from the group consisting of fully mineralizedelongate bone particles, fully demineralized elongate bone particles,substantially fully mineralized elongate bone particles, partiallydemineralized elongate bone particles, superficially demineralizedelongate bone particles, and mixtures thereof wherein mutuallycontacting elongate bone particles are entangled with each other;wherein the entangled bone particles form a coherent aggregate matrix,wherein the coherent aggregate matrix possesses an open pore, flexible,sponge-like structure configured to absorb fluids while generallyretaining predetermined dimensions; and wherein the osteoimplantcomprises a core composed of the fully demineralized elongate boneparticles which have not been compacted and an outer surface composed ofthe superficially demineralized elongate bone particles which have notbeen compacted surrounding the core.
 2. The osteoimplant of claim 1,wherein the osteoimplant comprises a continuous three-dimensionalstructure possessing visible pores.
 3. The osteoimplant of claim 1,wherein the osteoimplant comprises about 10-20 grams of the elongatedbone particles and the elongated bone particles occupy a volume of about100-120 cm³.
 4. The osteoimplant of claim 1, wherein the osteoimplant isconfigured to retain its shape once wetted.
 5. The osteoimplant of claim1, wherein the elongate bone particles represent at least about 50weight percent of the aggregate.
 6. The osteoimplant of claim 1, whereinthe elongate bone particles represent at least about 60 weight percentof the aggregate.
 7. The osteoimplant of claim 1, wherein the elongatebone particles represent at least about 90 weight percent of theaggregate.
 8. The osteoimplant of claim 1, wherein the aggregate furthercomprises non-elongate bone particles.
 9. The osteoimplant of claim 1,wherein the elongate bone particles are obtained from cortical,cancellous, corticocancellous allogenic, xenogenic or transgenic bonetissue.
 10. The osteoimplant of claim 1, wherein a median length of theelongate bone particles is from about 2 mm to about 200 mm, a medianwidth of the elongate bone particles is from about 0.05 mm to about 50mm and a ratio of median length to median width is from about 10:1 toabout 2000:1.
 11. The osteoimplant of claim 1, wherein a median lengthof the elongate bone particles is from about 1 mm to about 100 mm, amedian width of the elongate particles is from about 0.1 mm to about 20mm and a ratio of median length to median width is from about 20:1 toabout 600:1.
 12. The osteoimplant of claim 1, wherein the osteoimplantis swellable upon contact with body and/or irrigation fluids.
 13. Theosteoimplant of claim 1, wherein the osteoimplant has bulk density ofnot greater than about 0.3 g/cm³.
 14. An osteoimplant which compriseselongate bone particles selected from the group consisting ofsubstantially fully mineralized elongate bone particles, partiallydemineralized elongate bone particles, superficially demineralizedelongate bone particles, and mixtures thereof, wherein mutuallycontacting elongate bone particles are entangled with each other;wherein the entangled bone particles form a coherent aggregate matrix,wherein the coherent aggregate matrix possesses an open pore, flexible,sponge-like structure configured to absorb fluids while generallyretaining predetermined dimensions; and wherein the osteoimplantcomprises a core composed of the superficially demineralized elongatebone particles which have not been compacted and an outer surfacecomposed of the partially demineralized elongate bone particles whichhave not been compacted surrounding the core, wherein the non-compactedsuperficially demineralized elongate bone particles that make up thecore of the osteoimplant contain a higher percentage of their originalmineral content than do the non-compacted partially demineralizedelongate bone particles that make up the outer surface of theosteoimplant.
 15. An osteoimplant which comprises a carrier, a wettingagent and a coherent aggregate matrix of elongate bone particles havinga median length of from about 0.05 mm to about 200 mm and a ratio ofmedian length to median width of from about 10:1 to about 2000:1, theosteoimplant having the elongate bone particles distributed throughout,the elongate bone particles selected from the group consisting ofsubstantially fully mineralized elongate bone particles, partiallydemineralized elongate bone particles, superficially demineralizedelongate bone particles, fully demineralized elongate bone particles,and mixtures thereof, wherein mutually contacting elongate boneparticles are entangled with each other; wherein the aggregate matrixpossess an open pore, flexible, sponge-like structure and is configuredto absorb fluids while generally retaining a predetermined dimension;wherein the wetting agent comprises a liquid polyhydroxy compound; andwherein the osteoimplant comprises a core composed of the superficiallydemineralized elongate bone particles which have not been compacted andan outer surface composed of the fully demineralized elongate boneparticles which have not been compacted surrounding the core.
 16. Theosteoimplant of claim 15 wherein the elongate bone particles are admixedwith one or more components selected from the group consisting ofbinders/fillers, plasticizers, biostatic/biocidal agents, bioactivesubstances, surface active agents prior to, during and/or after formingthe aggregate into the osteoimplant.
 17. The osteoimplant of claim 16wherein the elongate bone particles are admixed with a binder/filler,and wherein the binder/filler is selected from the group consisting ofcyanoacrylates, epoxy-based compounds, dental resin sealants, dentalresin cements, calcium phosphate and calcium sulfate self-settingcements, glass ionosin cements, calcium phosphate and calcium sulfateself-setting cements, glass ionomer cements, polymethyl methacrylate,gelatin-resorcinol-formaldehyde glues, protein and collagen-based glues,acrylic resins, cellulosics, bioabsorbable polymers selected from thegroup consisting of polyglycolide, polylactide, glycolide-lactidecopolymers, polycaprolactone, polyanhydrides, polycarbonates,polyorthoesters, polyamino acids, polyarylates, polycyanoacrylates,polyhydroxybutyrate, polyhydroxyvalyrate, polyphosphazenes,polyvinylpyrrolidone, polyarylates, tyrosine-based polycarbonets andpolyarylates, carbohydrate polymers, polyiminocarbonates, polypropylenefumarates, polyanhydride esters, polytetrafluroethylene hexacryl,Hyaluronic acid, fibrin, fibrin-collagen, polyethylene glycol glues,mucopolysaccharides and mussell adhesive proteins, fatty acids and fattyacid derivatives.
 18. The osteoimplant of claim 16 wherein the elongatebone particles are admixed with a binder/filler, and wherein thebinder/filler is selected from the group consisting of bone powder,demineralized bone powder, porous calcium phosphate ceramics,hydroxyapatite, tricalcium phosphate, Bioglass® and other calciumphosphate materials, calcium sulfate or calcium carbonate particles. 19.The osteoimplant of claim 16 wherein the elongate bone particles areadmixed with a biostatic/biocidal agent, and wherein thebiostatic/biocidal agent is selected from the group consisting ofantibiotics, povidone, sugars and mucopolysaccharides.
 20. Theosteoimplant of claim 16 wherein the elongate bone particles are admixedwith a surface-active agent, and wherein the surface-active agent isselected from the group consisting of biocompatible nonionic, cationic,anionic and amphoteric surfactant.
 21. The osteoimplant of claim 16wherein the elongate bone particles are admixed with a bioactivesubstance, and wherein the bioactive substance is selected from thegroup consisting of trophic factors, analgesics, anti-cancer agents,vaccines, adjuvants, antibodies, neuroleptics, genes and geneticelements for transfection, cells or cellular components, collagen,insoluble collagen derivatives, and soluble solids and/or liquidsdissolved therein, antiviricides, particularly those effective againstHIV and hepatitis; antimicrobials and/or antibiotics selected from thegroup consisting of erythromycin, bacitracin, neomycin, penicillin,polymicin B, tetracyclines, biomycin, chloromycetin, and streptomycins,cephalosporins, ampicillin, azactam, tobramycin, clindamycin andgentamicin; biocidal/biostatic sugars selected from the group consistingof dextran, and glucose; amino acids, peptides, vitamins, inorganicelements, co-factors for protein synthesis; hormones; endocrine tissueor tissue fragments, synthesizers; enzymes selected from the groupconsisting of collagenase, peptidases, oxidases, polymer cell scaffoldswith parenchymal cells, angiogenic drugs and polymeric carrierscontaining such drugs; collagen lattices; antigenic agents; cytoskeletalagents; cartilage fragments, modified living cells selected from thegroup consisting of chondrocytes, bone marrow cells, mesenchymal stemcells, natural extracts, genetically engineered living cells orotherwise modified living cells, DNA delivered by plasmid or viralvectors, genes or genetic elements, tissue transplants, demineralizedbone powder, autogenous tissues, blood, serum, soft tissue, bone marrow,bioadhesives; non-collagenous proteins, osteopontin, osteonectin, bonesialo protein, laminin, fibrinogen, vitronectin, thrombospondin,proteoglycans, decorin, beta glycan, biglycan, aggrecan, versican,tenascin, matrix gla protein, hyaluronan, amino acids, amino acidresidues, peptides, bone morphogenic proteins (BMPs); osteoinductivefactor (OIF); fibronectin (FN); endothelial cell growth factor (ECGF);cementum attachment extracts (CAE); ketanserin; human growth hormone(HGH); animal growth hormones; epidermal growth factor (EGF);interleukin-1 (IL-1); human alpha thrombin; transforming growth factor(TGF-beta); insulin-like growth factor (IGF-1) (IGF-2); platelet derivedgrowth factors (PDGF); fibroblast growth factors (FGF, aFGF, bFGF,etc.); periodontal ligament chemotactic factor (PDLGF); somatotropin;bone digestors; antitumor agents; immuno-suppressants; fatty acids(including polar and non-polar fatty acids); permeation enhancers, fattyacid esters, laureate, myristate and stearate monoesters of polyethyleneglycol, enamine derivatives, alpha-keto-aldehydes, and nucleic acids;inorganic elements, inorganic compounds, cofactors for proteinsynthesis, hormones, soluble and insoluble components of the immunesystem; soluble and insoluble receptors including truncated forms;soluble, insoluble and cell surface bound ligands including truncatedforms; chemokines, bioactive compounds that are endocytosed; endocrinetissue or tissue fragments, growth factor binding proteins, insulin-likegrowth factor binding protein (IGFBP-2) (IGFBP-4) (IGFBP-5) (IGFBP-6);angiogenic agents, bone promoters, cytokines, interleukins, geneticmaterial, genes encoding bone promoting actions, cells containing genesencoding bone promoting action; growth hormones selected from the groupconsisting of somatotrophin; bone digestors; antitumor agents; cellularattractants and attachment agents; immunosuppressants; bone resorptioninhibitors and stimulators; angiogenic and mitogenic factors; bioactivefactors that inhibit and stimulate secondary messenger molecules; celladhesion molecules, cell-matrix and cell-cell adhesion molecules;secondary messengers, monoclonal antibodies specific to cell surfacedeterminants on mesenchymal stem cells, clotting factors; externallyexpanded autograft or xenograft cells, nucleic acids and any combinationthereof.
 22. The osteoimplant of claim 15 wherein the liquid polyhydroxycompound is glycerol.
 23. The osteoimplant of claim 15 wherein theelongate bone particles are in a lyophilized state.
 24. The osteoimplantof claim 15 wherein the elongate bone particles are cross-linked. 25.The osteoimplant of claim 15, wherein the osteoimplant is swellable uponcontact with body and/or irrigation fluids.
 26. The osteoimplant ofclaim 15, the osteoimplant having a shape selected from the groupconsisting of a sheet, plate, disk, cone, pin, screw, tube, tooth, toothroot, plug, bone or portion of bone, wedge or portion of wedge, cylinderor threaded cylinder.
 27. The osteoimplant of claim 15, the osteoimplantincluding at least one recess therein.
 28. The osteoimplant of claim 15wherein the osteoimplant is provided in at least one cavity of animplant for bone repair.
 29. The osteoimplant of claim 28 wherein theimplant for bone repair is an intervertebral implant.
 30. Theosteoimplant of claim 28 which the implant for bone repair is a fusioncage.
 31. The osteoimplant of claim 11 wherein the elongate boneparticles possess a ratio of median length to median width of from about20:1 to about 600:1.
 32. The osteoimplant of claim 15 wherein theosteoimplant is configured for use in posterolateral procedures.
 33. Theosteoimplant of claim 15, wherein the superficially demineralizedelongate bone particles include one or more regions of surface-exposedcollagen.
 34. The osteoimplant of claim 15, wherein the fullydemineralized elongate bone particles contain less than about 8% oftheir original mineral content.
 35. The osteoimplant of claim 15,wherein the mutually contacting elongate bone particles are mechanicallyentangled.
 36. The osteoimplant of claim 15, wherein the mutuallycontacting elongate bone particles are naturally entangled.
 37. Theosteoimplant of claim 15, wherein the elongate bone particles have amedian length of about 0.05 mm and a ratio of median length to medianwidth of about 10:1.
 38. The osteoimplant of claim 15, wherein thecoherent aggregate matrix has a bulk density of not greater than about0.3 g/cm³.
 39. The osteoimplant of claim 15, wherein the coherentaggregate matrix has a bulk density of approximately 0.114 g/cm³. 40.The osteoimplant of claim 15, wherein the coherent aggregate matrixoptionally includes bone particles selected from the group consisting ofsubstantially fully demineralized elongate bone particles, substantiallyfully demineralized non-elongate bone particles, substantially fullymineralized non-elongate bone particles, partially demineralizednon-elongate bone particles, superficially demineralized nonelongatebone particles and mixtures thereof.
 41. The osteoimplant of claim 15,wherein the osteoimplant comprises a compression strength of from about5 to about 200 MPa.
 42. The osteoimplant of claim 15, wherein theosteoimplant comprises a compression strength of from about 25 to about75 MPa.
 43. The osteoimplant of claim 15, wherein the osteoimplantincludes a concave depression configured to receive graft material. 44.The osteoimplant of claim 43, wherein the depression is a trough. 45.The osteoimplant of claim 44, further comprising sidewalls surroundingthe trough.
 46. The osteoimplant of claim 43, wherein the depression isperformed.
 47. The osteoimplant of claim 43, further comprisingsidewalls surrounding the concave depression.